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W. Zhang, M.F. Lou; The Presence of an Endogenous Superoxide Anion-Generating System in the Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):316.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose : Reactive oxygen species (ROS) has been implicated to have a physiological function in regulating and activating the mitogenic-associated signal transduction pathways in cells stimulated by certain growth factors or cytokines for various cellular functions. Arachidonic acid (AA) is known to be the immediate activator of the membrane NADPH oxidase in phagocytes and other cell types to generate superoxide. This study is to identify this ROS generating system in the lens epithelial cells using arachidonic acid (AA) as the stimulator. Methods : Confluent human lens epithelial cells (B3) (1.0 millions, serum free) were subjected to stimulation by AA, its metabolites and specific inhibitors to its metabolic enzymes. The generation of superoxide anion was quantified using lucigenin-amplified chemiluminescence (LUCL) in live HLE cells, which were trypsinized, resuspended in HEPES-buffered saline and mixed with lucigenin (5 mM). The LUCL readings were recorded with a luminometer (LumiStar BMG) immediately upon AA addition. Cells preloaded with superoxide dismutase (SOD) or mannitol were used as negative controls and cells mixed with 5% ethanol (solvent for AA) was used for baseline. A time- and concentration-dependent study on the AA-stimulated activation of mitogen activated protein kinases (JNK, MEK and ERK) were carried out using western blot analysis with respective phospho-specific antibobies. Results : AA at dosage of 30-150 µM proportionally induced luminescence generation in HLE cells, but was ineffective in cells preloaded with SOD or mannitol. Leinoleic acid showed a mild stimulatory effect, but stearic acid, eicosa-11Z,14Z,17Z-trienoic acid (20:3) and eicosa-11Z,14Z-dienoic acid (20:2) were ineffective. The generation of superoxide anion was not contributed by the prostaglandin or the cytochrome p450 (cyt-P450) pathway since indomethacin (inhibitor for cycloxygenase) or ketoconazole (inhibitor for cyt- P450) could not eradicate the stimulatory effect of AA. While CDC, a specific inhibitor to 12-HETE lipoxygenase, completely eliminated superoxide anion generation. 12-HETE, but not 5-leukitrienes or 5-HPETE, induced superoxide production 30-fold more effective than that of AA. Western blot analysis of the cell lysate showed that AA at concentrations of 30-150 µM progressively activated MEK, ERK and JNK. These signaling components were transiently activated between 2.5-30 min . Conclusions : A NADPH oxidase-associated ROS generating system is present in HLE cells and can be activated specifically by AA and its metabolite 12-HETE. Supported by NIH grant EY 10590. None
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